Reactivation of fetal globin expression in adult erythroid cells is a prized goal to ameliorate hemoglobinopathies such as Sickle Cell Disease and beta-thalassemia. We used human CD34+ erythroid progenitor cells to explore chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation during erythropoiesis. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this question. We targeted the mouse embryonic epsilon y gene in ES cells and then used recombinase mediated cassette exchange to replace it with epsilon y genes in which selected promoter transcription factor motifs had been destroyed or altered. Most mutations modestly perturbed epsilon y transcription, indicating that, individually, they are dispensable for LCR/epsilon communication. Substitution of the beta-globin CACCC site was a strong up-regulator of epsilon y transcription and resulted in recruitment of the adult specific factor EKLF to the promoter, consistent with an important role for the CACCC element in stage specific globin gene expression. In addition we inserted the cHS4 insulator between the LCR and epsilon y. The cHS4 insertion severely reduced transcription of embryonic y and beta-h1 genes and resulted in RNA pol II and non-genic transcript accumulation in the insulator. This observation has important implications for the mechanism by which LCR/enhancers and chromatin insulators work which we continue to explore. These experiments are novel since they alter transcription factor recruitment and chromatin organization in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo.